Resting T lymphocytes can be activated in vitro by monoclonal antibodies against the T cell receptor complex (TCR-CD3) or against CD2, a 50kD glycoprotein. Activation of resting T lymphocytes by means of monoclonal antibodies leads to proliferation and differentiation and therefore mimics the action of the naturally occurring ligands for those receptors (antigen for the T cell receptor or the LFA-3 for CD2). The earliest step of T cell activation by either monoclonal antibodies or the natural ligands is a phosphorylation of a limited number of intracellular and transmembrane proteins (e.g. CD3 epsilon, CD3 zeta, CD4, CD8, CD45). Phosphorylation of proteins is thought to be mediated by intracellular protein kinases which are activated upon the binding of monoclonal antibodies or the appropriate ligands and which phosphorylate proteins either on tyrosine residues (protein tyrosine kinases) or on threonine- and/or serine residues (serine/threonine kinases). Alternatively, it is possible that constitutive dephosphorylation could be inhibited by T cell activation and could therefore be responsible for the increased abundance of phosphoproteins observed. During the last ten years, an increasing number of protein kinases has been identified, but only a very limited number of those are tyrosine kinases. These protein tyrosine kinases can be divided into two groups:
a) Tyrosine kinases that are also integral membrane proteins with extracellular ligand binding domains and intracellular catalytic domains (e.g. EGF receptor, PDGF receptor, insulin receptor); and
b) Tyrosine kinases that do not possess an extracellular domain or membrane spanning region but associate with the inner leaf of the plasma membrane (e.g. p56.sup.lck, p60.sup.src, p59.sup.fyn).
Protein tyrosine kinases of both groups are encoded by protooncogenes and may therefore play a role in the origin of malignant cell growth. Recent studies have shown that protein tyrosine kinases play a key role in the regulation of cell growth and differentiation. Tyrosine kinases can themselves be phosphorylated on serine, threonine and tyrosine. Recent studies have shown that the enzymatic activity of certain tyrosine kinases is partially dependent upon the degree of phosphorylation on tyrosine. This leads us to the conclusion that the activity of tyrosine kinases is at least partially regulated by tyrosine phosphatases.
The CD45 molecule, an integral membrane tyrosine phosphatase, is expressed on all hematopoietic cells, and seems to play a very important regulatory role during an immune response. This is shown by a number or T cell functions that are either increased or inhibited by monoclonal antibodies against the CD45 protein. However, it is not known whether those antibodies modulate the enzymatic activity of the molecule or which proteins are the natural substrates for CD45. p56.sup.lck, a T cell specific tyrosine kinase that is associated with CD4, has been discussed as a possible substrate for CD45. However, no biochemical data are currently available that directly prove an association between CD45 and p56.sup.lck. Therefore, no substrates for CD45 have been definitively identified.
Using unconventional immunoprecipitation techniques we have identified a potential substrate of CD45 which is the subject of this patent. The molecule is an intracellular protein with a relative apparent molecular weight of 32kD (SDS-PAGE) and a pI of 4.0 to 4.5. In resting T cells, this protein, "pp32", is constitutively phosphorylated on serine. Immunoprecipitation experiments with anti-CD45 monoclonal antibodies have shown that pp32 is specifically associated with CD45. Besides pp32, a tyrosine kinase coprecipitates with the CD45 molecule. The coprecipitated protein kinase is responsible for in vitro phosphorylation of pp32 on tyrosine residues. The tyrosine kinase has been identified by immunoprecipitation and subsequent peptide analysis as p56.sup.lck. The ability of CD45 to use tyrosine phosphorylated pp32 as a substrate in vitro provides further evidence that an enzyme-substrate relationship exists for the two molecules in vivo. Detailed electrophoretic analysis or pp32 has subsequently shown that this protein exists (in resting T cells) in two isoforms (pp32 high and pp32 low). Both isoforms show rapid changes during the activation of T lymphocytes. The changes take place within 5 minutes after stimulation of resting T lymphocytes with monoclonaI antibodies specific for CD2 or with Phorbol esters.
Based upon the amino acid sequences of peptide fragments of the isolated pp32 protein, a nucleic acid molecule encoding pp32 has been isolated.